This invention relates to methods and systems for overlaying at least three microarray images to obtain a multicolor composite image.
Spots containing fluorescently labeled DNA samples on a suitable carrier such as a microscope slide or membrane are commonly known as microarrays or bio-chips. An example of a process that uses overlays is in the analysis and quantitation of such microarrays.
The microarrays are typically created with fluorescently labeled DNA samples in a grid pattern consisting of rows 22 and columns 20 typically spread across a 1 by 3 inch glass microscope slide 24, as illustrated in FIG. 1. The rows 22 extend along the smaller dimension of the slide 24 and the columns 20 extend along the larger dimension of the rectangular slide 24. Each spot 26 in the grid pattern (or array) 28 represents a separate DNA sample and constitutes a separate experiment. A plurality of such grid patterns 28 comprises an array set 30. Reference or xe2x80x9ctargetxe2x80x9d DNA (or RNA) is spotted onto the glass slide 24 and chemically bonded to its surface. Fluorescently labeled xe2x80x9cprobexe2x80x9d DNA (or RNA) is then introduced and allowed to hybridize with the target DNA. Excess probe DNA that does not bind is removed from the surface of the slide 24 in a subsequent washing process.
The purpose of the experiment is to measure the binding affinity between the probe and target DNA to determine the likeness of their molecular structures: complementary molecules have a much greater probability of binding than unrelated molecules. The probe DNA is labeled with fluorescent labels that emit light when excited by an external light source of the proper wavelength. The brightness of each sample on the slide 24 is a function of the fluor density in that sample. The fluor density is a function of the binding affinity or likeness of the probe molecule to the target molecule. Therefore, the brightness of each sample can be mapped to the degree of similarity between the probe DNA and the target DNA in that sample. On a typical microarray, up to tens of thousands of experiments can be performed simultaneously on the probe DNA, allowing for a detailed characterization of complex molecules.
Scanning laser fluorescence microscopes or microarray readers as illustrated in FIG. 2 can be used to acquire digital images of the emitted light from a microarray. The digital images are comprised of several thousand to hundreds of millions of pixels that typically range in size from 5 to 50 microns. Each pixel in the digital image is typically represented by a 16 bit integer, allowing for 65,535 different grayscale values. The microarray reader sequentially acquires the pixels from the scanned microarray and writes them into an image file which is stored on a computer hard drive. The microarrays can contain several different fluorescently tagged probe DNA samples at each spot location. The microarray scanner repeatedly scans the entire microarray with a laser of the appropriate wavelength to excite each of the probe DNA samples and store them in their separate image files. The image files are analyzed and subsequently viewed with the aid of a programmed computer.
A typical confocal laser microarray scanner or microarray reader is illustrated in FIG. 2. The reader is commonly used to scan the microarray slide 24 to produce one image for each dye used by sequentially scanning the microarray with a laser of a proper wavelength for the particular dye. Each dye has a known excitation spectra and a known emission spectra. The scanner includes a beam splitter 32 which reflects a laser beam 34 towards an objective lens 36 which, in turn, focuses the beam at the surface of slide 24 to cause fluorescence spherical emission. A portion of the emission travels back through the lens 36 and the beam splitter 32. After traveling through the beam splitter 32, the fluorescence beam is reflected by a mirror 38, travels through an emission filter 40, a focusing detector lens 42 and a central pinhole 44.
Analysis and Quantitation of the Microarray in Two Colors
Analysis of the fluor density at each spot location requires software algorithms that utilize image processing algorithms to locate all the spots and measure the brightness of the pixels in each spot. Visual feedback of the relative spot intensities has commonly been done using software to overlay the two images by encoding the brightness of the image as a function of the brightness of one color for each image, i.e. red or green. When red and green objects are overlayed at the same pixel location, a yellow color is produced as illustrated in FIG. 3.
Given
B1 is the brightness of a pixel in image 1,
B2 is the brightness of a pixel in image 2,
each brightness value is from 0 to 65,535,
Image 1 is to be overlayed in red and an 8 bit pixel will be R1,
Image 2 is to be overlayed in green and an 8 bit pixel will be G1.
R1=B1/256
G1=B2/256
A common method for displaying pixels on a computer monitor or printout is in true color, comprised of 24 bits per pixel, where 8 bits each represent the red, green, and blue color channels, respectively.
Pixel (R, G, B)=R1, G1, 0
The visual display of the overlayed images provides the viewer with feedback on the brightness of each spot from each channel of the reader, the relative brightness of each spot to the others by the degree of the color yellow present, and a measure of the spatial registration between images by the amount of red or green on either side of a central yellow color as illustrated in FIG. 3.
The use of two color overlays is common and has been demonstrated by BioDiscovery in a software program called ImaGene and by Stanford University in a software program called ScanAlyze, as well as several places in the literature. Two color visualization has been designed to meet the needs of two color microarray experiments. Two color microarray readers have been designed and built by several companies and are readily available in the market. Microarray readers with more than two colors have just recently been released by the assignee of the present application.
One common use of overlays in microarray experiments and quantitation is to provide visual feedback to scientists of any mis-registration between images. Many microarray readers sequentially scan the microarray with one scan for each DNA probe used. A scan typically starts in the upper left corner of the microarray, usually called the origin, and proceeds in a left to right and top to bottom direction until the entire area has been scanned. When a scan has finished, the scanner mechanically resets the laser to the origin in preparation for the next scan. The difference in the actual position between each image scan is a mis-registration error. By viewing the overlayed image and by zooming the spot features, a scientist can visually quantify the amount of mis-registration between the images.
Another common use of the overlays in microarray experiments is to correct for any mis-registration errors. Typically, the images are presented in a single viewing window with each image assigned a different color. In a two color system, this would typically be green and red. The locations where a match is obtained will be displayed in yellow. The scientist can visually see the areas of green and red on the fringes of a microarray spot and, using a keyboard or mouse, move one image relative to the other until the fringe areas turn yellow. The best registration match will minimize the green and red fringes across the entire microarray pattern.
Articles related to the present invention include the following:
Brown, A. J., et al. xe2x80x9cTargeted Display: A New Technique for the Analysis of Differential Gene Expressionxe2x80x9d, METHODS ENZYMOL. 1999; 303:392-408;
Eisen, M. B., et al. xe2x80x9cDNA Arrays for Analysis of Gene Expressionxe2x80x9d, METHODS ENZYMOL. 1999; 303:179-205;
Zhang, H., et al. xe2x80x9cDifferential Screening of Gene Expression Difference Enriched By Differential Displayxe2x80x9d, NUCLEIC ACIDS RES. Jun. 15, 1996;24(12):2454-5;
Adryan, B., et al. xe2x80x9cDigital Image Processing For Rapid Analysis of Differentially Expressed Transcripts on High-Density cDNA Arraysxe2x80x9d, BIOTECHNIQUES. Jun. 26, 1999(6):1174-9;
Wadler, S., et al. xe2x80x9cQuantification of Ribonucleotide Reductase Expression in Wild-Type and Hydroxyurea-Resistant Cell Lines Employing In Situ Reverse Transcriptase Polymerase Chain Reaction and a Computerized Image Analysis Systemxe2x80x9d, ANAL. BIOCHEM. Feb. 1, 1999;267(1):24-9; and
Zhao, N., et al. xe2x80x9ccDNA Filter Analysis: A Novel Approach For Large-Scale, Quantitative Analysis of Gene Expressionxe2x80x9d, GENE. Apr. 24, 1995; 156(2):207-13.
An object of the present invention is to provide a method and system for overlaying at least three microarray images to obtain a multicolor composite image.
In carrying out the above objects and other objects of the present invention, a method is provided for overlaying at least three microarray images to obtain a multicolor composite image. The method includes providing at least three microarray images wherein each of the images includes a plurality of pixels having brightness values. The method also includes assigning each image a different color to obtain colorized images and combining corresponding pixels of each colorized image to obtain overlayed images. The overlayed images form a multicolored composite image. The method also includes the step of displaying the multicolored composite image.
When the overlayed images have a registration, the method may include the step of processing the overlayed images to alter the registration. The step of processing may include the step of receiving a command to move one of the overlayed images relative to the other overlayed images.
The step of providing may include the step of scanning a microarray at at least three different wavelengths to obtain the microarray images.
The step of assigning may include the step of creating a palette having entries for each of the wavelengths to display the microarray images. The entries for each palette are based on a composite color and the brightness values of the pixels of its respective image.
Also, preferably, the step of combining includes the step of logically ORing corresponding pixels of each colorized image.
Still, preferably, the method includes the step of receiving at least one command to select a composite color for each microarray image to be overlayed.
Still further in carrying out the above objects and other objects of the present invention, a system is provided for carrying out each of the above method steps.
The above objects and other objects, features, and advantages of the present invention are readily apparent from the following detailed description of the best mode for carrying out the invention when taken in connection with the accompanying drawings.